We have been investigating the phosphorylation of smooth muscle and nonmuscle myosin by protein kinase C (PKC). Protein kinase C phosphorylates the smooth muscle myosin light chain at two sites; serine-1 or 2 and threonine-9. Threonine-9 is preferentially phosphorylated at a rate approximately 2 - 3 times faster than that of serine-1 or 2. Two-dimensional tryptic peptide mapping of the 20kDa light chain of platelet myosin phosphorylated by PKC demonstrates two phosphorpeptides that are similar to those seen following digestion of the smooth muscle myosin light chain. When intact human platelets are stimulated with phorbol myristate acetate, however, there is only one major phosphopeptide seen on two-dimensional tryptic peptide mapping. This corresponds to the serine site seen in the peptide map of the in vitro phosphorylated light chain. In vitro studies indicate that this in vivo specificity is dependent on ionic strength. In addition, we have demonstrated that protein kinase C phosphorylates the myosin heavy chain in vitro, using human platelet myosin. Heavy chain phosphorylation results in a single major tryptic phosphopeptide that contains phosphoserine and has been localized to the LMM portion of the heavy chain by limited chymotryptic digestions. To expedite two-dimensional peptide mapping of the phophorylated light chain from in vivo systems, we have recently started to map peptides of the phosphorylated light chain using HPLC.